Location: Foreign Arthropod Borne Animal Disease Research
2023 Annual Report
Objectives
Objective 1: Identify factors associated with Rift Valley Fever virus infections, pathogenesis and maintenance in vector and animal hosts.
• Characterize host, vector and bunyavirus interactions (molecular and cellular) associated with virus infection.
• Identify epidemiological and ecological factors affecting the inter-epidemic cycle.
• Develop means to detect and characterize emergent arboviral diseases and use these data to generate models that predict future outbreaks.
Sub-objective 1.A: Identify cellular factors important for replication and pathogenesis in the vector and the mammalian host cells.
Sub-objective 1.B: Examine the effects of temperature on the extrinsic incubation period of RVFV in various mosquito species.
Sub-objective 1.C.: Develop deployable tools for rapid diagnosis and characterization of RFVF.
Approach
Rift Valley fever (RVF) is a long-recognized disease of domestic livestock in Africa caused by the arthropod-borne virus, Rift Valley Fever virus (RVFV). RVFV is a zoonotic pathogen present on the World Organisation for Animal Health (OIE) list of notifiable animal diseases of concern and the World Health Organization priority disease list. RVFV is a significant threat to U.S. livestock due to the presence of naïve host populations and competent vectors. RVFV transmission is dependent on complex interactions involving virus, arthropod vector, mammalian host, and environment. Despite the importance of this disease, many elements for RVFV replication, pathogenesis, transmission, and disease epidemiology are poorly understood. The objective of this study is to identify factors associated with RVFV infections, pathogenesis and maintenance in vector and animal hosts. This objective is split into three parts: 1) characterize host, vector and bunyavirus interactions associated with virus infection, 2) identify epidemiological and ecological factors affecting the inter-epidemic cycle and 3) develop means to detect and characterize emergent arboviral diseases and use these data to generate models that predict future outbreaks. The objective is addressed by the following sub-objectives. Sub-objective 1A identifies cellular factors important for virus replication and pathogenesis in disparate hosts by examining how Rho GTPases effects RVFV replication. Rho GTPases are key regulators of cytoskeleton rearrangement and play an important role in virus replication. Effects of temperature on RVFV extrinsic incubation period in two mosquito species will be examined for Sub-objective 1B. These studies will ascertain the potential of a mosquito species to maintain and transmit RVFV during the various temperatures associated with inter-epidemic periods. Sub-objective 1C will develop deployable diagnostic and next-generation sequencing assays to rapidly detect and characterize RVFV. These tools will provide data on virus emergence and will assist in surveillance, development of risk assessments, and predictive modeling.
Progress Report
Rift Valley Fever virus is a mosquito-borne virus that is transmitted to livestock through the bite of an infected mosquito. Little is known about the virus-vector and virus-animal interactions important for the transmission and survival of the virus in nature. Studies were performed to examine Objective 1: Identify factors associated with Rift Valley Fever virus (RVFV) infections, pathogenesis and maintenance in vector and animal hosts. Progress was made on the characterization of host, vector and bunyavirus interactions (molecular and cellular) associated with virus infection by detecting several Rho-family GTPases and confirming cross-reactivity of the GTPase antibodies in insect cell lines. Rho-family GTPases are molecules that play an important role in cell migration and remodeling of the cell structure. The development of protocols to examine the activation and inhibition of the Rho-family GTPases were initiated. Once developed, these protocols will be utilized to understand the role of the Rho-family GTPAses during virus replication and the diverse pathogenic effects seen in the insect versus mammalian cell lines. In addition, studies have demonstrated that virus produced in mosquito cells consistently induce more clinical responses compared to virus produced in mammalian cells. This may be due in part to structural difference in the virus particles produced in the disparate cell lines (mammalian and insect). Through collaboration with researchers at the Canadian Food Inspection Agency, studies were initiated to examine host effects on virus packaging. Over the past year, the protocol to standardize growth for optimal amplification of RVFV in cell lines derived from different mammalian and insect species were initiated. This standardization will allow for consistent results when comparing the role of virus packaging on the transmission and replication of the virus.
Through collaboration with researchers at Colorado State University, the effects of the environment on vector-borne disease virus replication transmission in North American mosquito species is being examined. Samples have been collected from one experimental replicate of mosquitoes exposed to RVFV at different temperatures. Data analysis that will provide fundamental information for predicting virus epidemiology and ecological factors affecting virus transmission is ongoing.
Early detection of RVFV is critical for a rapid response and to effectively control the associated disease. Thus, artificial genetic material was used to develop a protocol for point-of-need detection of RVFV. This protocol was further tested using genetic material collected from RVFV infected cells. Once fully developed, this technology will allow for rapid detection of RVFV that can be easily used in areas lacking easy access to testing facilities.
Accomplishments
1. Established a Culex tarsalis (CxTr) cell line for arbovirus research. ARS scientists in Manhattan, Kansas, established a cell line from Culex tarsalis eggs that provides a research tool for arbovirology. The cell line was confirmed as Cx. tarsalis by sequencing and was found to be free of contaminating cells, bacteria, fungi, and mycoplasma. The suceptability of CxTr cells to arbovirus infection was investigated with vaccine and wildtype arboviruses from four viral families: Flaviviridae (Japanese encephalitis virus), Phenuiviridae (Rift Valley fever phlebovirus), Rhabdoviridae (vesicular stomatitis virus), and Togaviridae (Mayaro virus). All viruses were able to infect and replicate within CxTr cells. This provides a valuable research tool for arboviriology and has already been shared with three other arbovirology research groups.
2. Tool for rapid genetic screening of Rift Valley fever virus (RVFV) genotypes. Mosquito borne Rift Valley fever phlebovirus (RVFV) is an emerging, zoonotic pathogen capable of exchanging gene segments between RVFV strains and potentially other related viruses. To investigate the ability of this phenomenon to occur under various conditions, genotyping (GT) assays were developed by ARS scientists in Manhattan, Kansas. The GT amplification assay produces genetic replicates with unique melting temperatures that are resolved in a post amplification melt curve analysis for strain identification. The assays are useful as screening tools for determining reassortment of the segmented RVFV genome during coinfections and could be adapted and applied for other segmented pathogens of interest.
3. Identified host factors that affect Rift Valley fever virus (RVFV) replication. Rift Valley fever is a zoonotic disease of high concern because the causative mosquito-borne virus infects both animals and humans. There are currently no control measures available to prevent the spread of RVFV in non-endemic countries. Endemic countries have some veterinary vaccines available, but they are often not deployed except during an outbreak. Like other viruses, the host’s cellular system is required to complete the RVFV multiplication cycle. Therefore, one way to control virus spread is to interrupt the interaction between the virus and its host. ARS scientists in Manhattan, Kansas, and collaborators found one important virus host factor that supported Rift Valley fever virus multiplication. The identification of this host factor is important for the scientific community as it can potentially lead to the development of antiviral strategies to control Rift Valley fever in both humans and animals.
4. Development of environmentally friendly insecticidal nanoparticles. The particle construction process using core and coating technology enabled the development of highly efficacious particles that kill mosquito larvae at 100 parts per billion concentrations. ARS scientist in Manhattan, Kansas, in collaboration with Kansas State University in Manhattan, Kansas, developed biodegradable particles using zein (corn) based cores coated with insecticidal silver particles, which are 10 to 100 times more lethal than existing silver nanoparticles. The zein particles are edible and nutritious therefore they are actively foraged and consumed by the mosquito larvae. Once consumed, the antimicrobial and antifungal characteristics of the silver coating kill the gut fauna of the mosquitoes resulting in death or failure to develop into adults. Particle construction is an environmentally friendly process allowing for wide area use of the particles and without caustic or corrosive reducing catalysts, the particle fabrication is significantly more economical and easier to scale up than existing particles. Furthermore, the particles' cores can be produced using recycled (upcycled) waste from food processing by-products. The core and coating technology can be used with existing pesticides or novel active ingredients to make new insecticides that take advantage of the mosquito larvae’s natural foraging behaviors to combat the evolution of pesticide resistance.
Review Publications
Shults, P.T., Eyer, P., Moran, M., Richardson, S., Liu, K., Blumenfeld, A., Davis, R., Vargo, E. 2023. Comparative genetic study of the colony structure and colony spatial distribution between the higher termite Amitermes parvulus and the lower, subterranean termite Reticulitermes flavipes in an urban environment. Insectes Sociaux. https://doi.org/10.1007/s00040-023-00919-2.
Balaraman, V., Gaudreault, N.N., Trujillo, J.D., Indran, S.V., Wilson, W.C., Richt, J.A. 2023. RT-qPCR genotyping assays for differentiating Rift Valley fever phlebovirus strains. Journal of Virological Methods. 315. Article 114693. https://doi.org/10.1016/j.jviromet.2023.114693.
Shults, P.T., Cohnstaedt, L.W., Zhang, X., Gerry, A., Eyer, P., Vargo, E. 2023. Immigration and seasonal bottlenecks: High inbreeding despite high genetic diversity in an oscillating population of Culicoides sonorensis (Diptera: Ceratopogonidae). Journal of Medical Entomology. https://doi.org/10.1093/jme/tjad068.
